Journal: Frontiers in Immunology

Article Title: Astrocyte-derived exosomal miR-378a-5p mitigates cerebral ischemic neuroinflammation by modulating NLRP3-mediated pyroptosis

doi: 10.3389/fimmu.2024.1454116

Figure Lengend Snippet: Astrocyte-derived exosomes enhanced brain injury recovery and attenuated pyroptosis in rats subjected to MCAO. (A, B) Fluorescence imaging revealed the entry of PKH26-labeled astrocyte-derived exosomes into the brain. Scale bar = 25 µm. (C, D) Nissl staining was performed to assess neuronal survival. (E, F) Immunofluorescence staining demonstrated the expression of GSDMD, a key protein involved in pyroptosis, in cortical neurons on the ischemic side of rats. Scale bar = 50 µm. (G-J) The amounts of GSDMD, Caspase-1 p20, and NRP3 proteins in the cortical region on the ischemic side were assessed by Western blot analysis. The statistical data for every group is presented as the mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01.

Article Snippet: After separation by 12% gel electrophoresis, the membrane was transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk powder for 2 h. The membrane was then incubated overnight at 4°C with primary antibodies against CD81 (1:1000, ab109201, Abcam), TSG101 (1:1000, ab125011, Abcam), GAPDH (1:10,000, 81649-5-RR, Proteintech), GSDMD (1:1000, AF4012, Affinity), NLRP3 (1:1000, PAB38738, Bioswamp), Caspase-1 p20 (1:1000, AF4005, Affinity), and β-actin (1:2000, 20536-1-AP, Proteintech).

Techniques: Derivative Assay, Fluorescence, Imaging, Labeling, Staining, Immunofluorescence, Expressing, Western Blot

Journal: Aging Cell

Article Title: Amyloid‐β activates NLRP3 inflammasomes by affecting microglial immunometabolism through the Syk‐AMPK pathway

doi: 10.1111/acel.13623

Figure Lengend Snippet: Aβ42 activates NLRP3 inflammasome in LPS‐primed microglia. Primary microglia were untreated (Veh) or primed with LPS (10 ng/ml, 3 h), followed by Aβ42 (4 μM) for 24 h. (a) Immunoblot showed that the protein expression levels of pro‐IL‐1β, pro‐IL‐18, and caspase‐1 (p20) were elevated after adding Aβ42 in LPS‐primed microglia. (b–d) Quantification of pro‐IL‐1β (b), pro‐IL‐18 (c) and caspase‐1 (d). (e) Representative immunofluorescence image of primary microglia with anti‐caspase‐1 (green). DAPI represents the nuclear signal (blue). Scale bar: 20 μm. (f) Quantification of immunofluorescence of caspase‐1. (g–i) The mRNA gene expression of NLRP3 (g), IL‐1β (h), and IL‐18 (i) was measured by real‐time PCR and expressed as relative fold change to vehicle group. (j) Immunofluorescence staining for ASC (red) speck. Scale bar: 20 μm. (k) Percentages of microglia containing ASC foci were quantified. (l) Immunoblot analysis of indicated proteins in conditioned media. Proteins from equal initial volumes were precipitated by TCA, and the levels of secreted proteins were quantified. (m–p) Quantification of secreted ASC (m), cleaved caspase‐1 (n), mature IL‐1β (o), and mature IL‐18 (p). (q) Immunoblot analysis of signaling proteins from cell lysates. (r–t) Quantitative analysis showed relative level of pSyk/Syk (r), pAMPK/AMPK (s), and pAkt/Akt (t). All data are shown as means ± SEM from four independent experiments and are analyzed by unpaired Student's t test. Confocal imaging data are from 5 randomly chosen fields per coverslip (e) or at least 200 cells per group (j). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: The primary antibodies used for immunoblotting were NLRP3 (1:1000, AG‐20B‐0014, Adipogen, San Diego, USA), IL‐1β (1:1000, AF‐401‐NA, R&D Systems, Minnesota, USA), IL‐18 (1:2000, 5180R‐100, BioVision, Milpitas, USA), caspase‐1 p20 (1:1000, AG‐20B‐0042‐C100, Adipogen), pAMPK (1:1000, 2535, Cell Signaling), AMPK (1:1000, 2532, Cell Signaling), pAkt (1:1000, 92715, Cell Signaling), Akt (1:1000, 9272, Cell Signaling), pSyk (1:1000, MA5‐14918, Thermo Fisher Scientific), Syk (1:1000, MA1‐19332, Thermo Fisher Scientific), pASC (1:1000, AP5631, ECM Biosciences, Kentucky, USA), ASC (1:1000, NBP1‐78977, Novus Biologicals, USA), Mfn2 (1:500, sc‐100560, Santa Cruz, Texas, USA), Drp1 (1:500, sc‐101270, Santa Cruz), and β‐actin (3700S, Cell Signaling). β‐Actin was used as a loading control.

Techniques: Western Blot, Expressing, Immunofluorescence, Gene Expression, Real-time Polymerase Chain Reaction, Staining, Imaging

Journal: Aging Cell

Article Title: Amyloid‐β activates NLRP3 inflammasomes by affecting microglial immunometabolism through the Syk‐AMPK pathway

doi: 10.1111/acel.13623

Figure Lengend Snippet: Inhibition of Syk protects Aβ42‐induced mitochondrial fission in LPS‐primed microglia. LPS‐primed microglia were pretreated with Syk inhibitor Bay 61‐3606 for 1 h followed by treatment with Aβ42 for 24 h. (a) Representative immunoblot of the protein levels of Drp1, Mfn2, and β‐actin in total cell lysate. (b and c) Densitometric quantifications showing the Drp1 (b) and Mfn2 (c). β‐Actin was used as a loading control. (d) Representative images of mitochondrial morphology, revealed by Tom20 staining. Mitochondria and nuclei were stained with Tom20 (red) and DAPI (blue), respectively. Scale bar: 10 μm. (e) Quantification of mitochondrial length. (f) Level of ROS in microglia stained with MitoSOX (red) and DAPI (blue) staining. Scale bar: 10 μm. (g) MitoSOX fluorescence intensity relative to veh was quantified. (h–k) LPS‐primed microglia were pretreated with Mdivi‐1, a selective mitochondria division inhibitor, for 1 h followed by treatment with Aβ42 for 24 h. pro‐IL‐1β (i), pro‐IL‐18 (j), caspase‐1 (p20) (k), and β‐actin levels were determined and quantified by Western blot. β‐Actin was used as a loading control. Data are shown as mean ± SEM from four independent experiments and are analyzed by one‐way ANOVA and Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: The primary antibodies used for immunoblotting were NLRP3 (1:1000, AG‐20B‐0014, Adipogen, San Diego, USA), IL‐1β (1:1000, AF‐401‐NA, R&D Systems, Minnesota, USA), IL‐18 (1:2000, 5180R‐100, BioVision, Milpitas, USA), caspase‐1 p20 (1:1000, AG‐20B‐0042‐C100, Adipogen), pAMPK (1:1000, 2535, Cell Signaling), AMPK (1:1000, 2532, Cell Signaling), pAkt (1:1000, 92715, Cell Signaling), Akt (1:1000, 9272, Cell Signaling), pSyk (1:1000, MA5‐14918, Thermo Fisher Scientific), Syk (1:1000, MA1‐19332, Thermo Fisher Scientific), pASC (1:1000, AP5631, ECM Biosciences, Kentucky, USA), ASC (1:1000, NBP1‐78977, Novus Biologicals, USA), Mfn2 (1:500, sc‐100560, Santa Cruz, Texas, USA), Drp1 (1:500, sc‐101270, Santa Cruz), and β‐actin (3700S, Cell Signaling). β‐Actin was used as a loading control.

Techniques: Inhibition, Western Blot, Control, Staining, Fluorescence

Journal: Aging Cell

Article Title: Amyloid‐β activates NLRP3 inflammasomes by affecting microglial immunometabolism through the Syk‐AMPK pathway

doi: 10.1111/acel.13623

Figure Lengend Snippet: Flufenamic acid inhibits Aβ42‐induced inflammasome activation in LPS‐primed microglia. Microglia were primed with LPS (10 ng/ml) for 3 h, and stimulated with Aβ42 (4 μM) for 24 h or treated with FA (200 μM) for 1 h before Aβ42 exposure. (a) Immunocytochemical analysis of activated caspase‐1 (green) and DAPI staining (blue). Scale bar: 20 μm. (b) Quantification of the fluorescence intensity of caspase‐1. (c) Immunocytochemical analysis of ASC speck formation. Scale bar: 10 μm. (d) Percentage of cells containing ASC speck. (e) Cell culture supernatants were assayed for murine IL‐1β by ELISA. (f–h) Quantitative real‐time PCR analysis of NLRP3 (f), IL‐18 (g), and IL‐1β (h) mRNA expression. (i–m) Proteins in cultured medium were collected by TCA precipitation. Caspase‐1 (p20) (j), ASC (k), IL‐1β (l), and IL‐18 (m) were determined and quantified by immunoblotting. (n–q) Immunoblot analysis and quantification by densitometry of pSyk, total Syk, pAMPK, total AMPK, pAkt, total Akt, and β‐actin. (r–t) Mitochondrial fission‐ and fusion‐related molecules were measured by immunoblotting in LPS‐primed Aβ‐treated microglia with or without FA. (u) Relative phagocytic activity of microglia measured by bead uptake assay. Data are presented as mean ± SEM from minimum of two independent experiments, and data were analyzed by one‐way ANOVA and Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: The primary antibodies used for immunoblotting were NLRP3 (1:1000, AG‐20B‐0014, Adipogen, San Diego, USA), IL‐1β (1:1000, AF‐401‐NA, R&D Systems, Minnesota, USA), IL‐18 (1:2000, 5180R‐100, BioVision, Milpitas, USA), caspase‐1 p20 (1:1000, AG‐20B‐0042‐C100, Adipogen), pAMPK (1:1000, 2535, Cell Signaling), AMPK (1:1000, 2532, Cell Signaling), pAkt (1:1000, 92715, Cell Signaling), Akt (1:1000, 9272, Cell Signaling), pSyk (1:1000, MA5‐14918, Thermo Fisher Scientific), Syk (1:1000, MA1‐19332, Thermo Fisher Scientific), pASC (1:1000, AP5631, ECM Biosciences, Kentucky, USA), ASC (1:1000, NBP1‐78977, Novus Biologicals, USA), Mfn2 (1:500, sc‐100560, Santa Cruz, Texas, USA), Drp1 (1:500, sc‐101270, Santa Cruz), and β‐actin (3700S, Cell Signaling). β‐Actin was used as a loading control.

Techniques: Activation Assay, Staining, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, TCA Precipitation, Western Blot, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Translocation of gasdermin D induced mitochondrial injury and mitophagy mediated quality control in lipopolysaccharide related cardiomyocyte injury

doi: 10.1002/ctm2.1002

Figure Lengend Snippet: Dynamic changes of mitophagy and its interaction with mitochondria localized GSDMD‐N at protein level. (A) Isolated mitochondrial fraction and cytosolic fraction were verified by detection of cytosolic specific marker GAPDH and mitochondrial specific marker COX IV. Full length GSDMD, GSDMD‐N fragment, full‐length EYFP‐GSDMD‐GFP fusion protein, and GSDMD‐N‐GFP were detected in the separated fraction of whole cell lysate and mitochondrial fraction, respectively; (B) level changes (corrected by GAPDH level) of Pink‐1, Parkin, p62, LC3B II/I ratio, Caspase‐1 p20 and GSDMD‐N fragment after 1 h (*: p < .01, comparing with control), 3 h (#: p < .01, comparing with control), and 6 h (†: p < .01, comparing with control) of Nigericin (5 μM) intervention following LPS (1 mg/L) 3 h treatment in wild‐type HL‐1 cells ( p < .01 by ANOVA, comparing among all groups); (C) level changes (corrected by GAPDH level) of Pink‐1, Parkin, p62, LC3B II/I ratio, Caspase‐1 p20 and GSDMD‐N fragment after 1 h (*: p < .01, comparing with control), 3 h (#: p < .01, comparing with control), and 6 h (†: p < .01, comparing with control) of Nigericin intervention following LPS 3 h treatment in GSDMD‐KO HL‐1 cells ( p < .01 by ANOVA, comparing among all groups); (D) considering no GSDMD‐N fragments in saline treated HL‐1 cells, LPS 3 h plus Nigericin 1 h or Nigericin 2 h partly inducing GSDMD fragmentation, and LPS 3 h plus Nigericin 3 h completely inducing GSDMD fragmentation, LPS 3 h plus Nigericin 3 h was chosen as the intervention protocol for in‐input experiment for CO‐IP. In the step of input experiment, both full‐length GSDMD and its cleaved N‐terminal fragment were pulled down by themselves under induced inflammatory stress mentioned above. Then, on the one hand, LC3B was detected in GSDMD‐N combined fractions under inflammatory stress, indicating direct or indirect combination between LC3B and GSDMD. Finally, on the other hand, both GSDMD and its N‐terminal fragment was detected in LC3B combined fractions, which confirm the combination of LC3B and GSDMD

Article Snippet: Then, membranes were incubated with primary antibodies of GSDMD (1:1000, ab209845, Abcam), cleaved Caspase‐1 p20 (1:1000, 4199, CST), LC3B (1:1000, 2775, CST), sequestosome‐1 (SQSTM1/p62) (1:1000, 5114, CST), COX‐4 (1:1000, CST), cleaved GSDMD‐N (1:1000, Abcam, EPR20829‐408), β‐Actin (1:5000, 12620, CST), GAPDH (1:5000, 8884, CST), Parkin (1:1000, 4211, CST), or Pink1 (1:1000, ab23707, abcam) at 4°C overnight.

Techniques: Isolation, Marker, Control, Saline, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Gut Microbiota Dysbiosis-Mediated Gut NLRP3 Inflammasome Activation Exacerbates Corticospinal Tract Injury After Intracerebral Hemorrhage

doi: 10.64898/2026.02.23.707330

Figure Lengend Snippet: (A) Experimental timeline and methods. Created with BioRender. (B) ICH model validation. (C, F) Representative immunofluorescence images showing MBP and NF200 expression in the cervical spinal cord (n=4/group). Scale bar: 50 μm. Statistical analysis by the two-tailed t test. (D-E, G-H) Representative images of Iba-1 and GFAP immunostaining in the cervical spinal cord at 14 days post-ICH (n=4/group). Scale bar: 100 μm. Statistical analysis by the two-tailed t test. (I, L) Representative immunofluorescence images showing MBP and NF200 expression in the lumbar spinal cord (n=4-5/group). Scale bar: 50 μm. Statistical analysis by the two-tailed t test. (J-K, M-N) Representative images of Iba-1 and GFAP immunostaining in the lumbar spinal cord at 14 days post-ICH (n=3-5/group). Scale bar: 100 μm. Statistical analysis by the two-tailed t test. (O) Representative H&E-stained sections of colon tissue at 3, 7 and 14 days post-ICH. Scale bar: 200 μm. (P) Western blot analysis of tight junction proteins (Occludin, ZO-1) in colon tissue. β-actin served as the loading control, n=5/group. Statistical analysis by the two-tailed t test. (Q) Western blot analysis of NLRP3, Caspase-1 p20, and IL-1β in colon tissue. β-Tubulin served as the loading control, n=5/group. Statistical analysis by two-tailed t test, except for the Caspase-1 p20 protein data, was analyzed by Welch’s t test. (R-S) ELISA measuring levels of Caspase-1 and IL-1β from peripheral blood, n=3-5/group. Statistical analysis by the two-tailed t test. (T) Assessment of microbiota α-diversity, n=8-9/group. The InvSimpson index was compared using a two-tailed Welch’s t-test, while other α-diversity indices were compared using a two-tailed Student’s t-test. (U)Principal Coordinate Analysis (PCoA) based on unweighted Bray-Curtis Distance, n=8-9/group (permutational multivariate analysis of variance). (V-W) Linear discriminant analysis Effect Size (LEfSe) identifying differentially abundant bacterial taxa, n=8-9/group (Kruskal-Wallis test). Data are presented as mean ± SEM. Significance levels are defined as *p < 0.05, **p < 0.01. C, contralateral; I, ipsilateral; ns, no significance.

Article Snippet: The membranes were blocked at room temperature in 5% non-fat milk for 1 hour, then incubated overnight at 4 □ with following primary antibodies: rabbit anti-NLRP3 (1:1000, immunoway, YM8024), rabbit anti-cleaved Caspase-1 p20 (1:1000, abmart, P79884R2M), rabbit anti-IL-1β (1:1000, MCE, HY-P80720), rabbit anti-ZO-1 (1:5000, proteintech, 21773-1-AP), rabbit anti-Occludin (1:5000, proteintech, 27260-1-AP), rabbit anti-β-tubulin (1:5000, affinity, AF7011), and rabbit anti-actin (1:5000, Beyotime, AF5006).

Techniques: Biomarker Discovery, Immunofluorescence, Expressing, Two Tailed Test, Immunostaining, Staining, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Gut Microbiota Dysbiosis-Mediated Gut NLRP3 Inflammasome Activation Exacerbates Corticospinal Tract Injury After Intracerebral Hemorrhage

doi: 10.64898/2026.02.23.707330

Figure Lengend Snippet: (A) Schematic diagram of the experimental timeline and treatment groups, created with BioRender ( https://BioRender.com/s5rgq47 ). (B, C) Western blot analysis of NLRP3 inflammasome components (NLRP3, Caspase-1 p20, IL-1β) in colon tissues from the indicated groups, n=4/group. Statistical analysis by the two-tailed t test. (D, F) Representative open-field test images and quantification of total travel distance in the open-field test across timepoints at 1, 3, 7, 14 days (n=9 mice/group). Statistical analysis by two-way ANOVA and Sidak’s multiple comparisons test. (E, G) Representative paw print patterns from the CatWalk XT gait analysis system, and the regularity index from each group at 3, 7 and 14 days. n=4 mice/group. Statistical analysis by two-way ANOVA and Sidak’s multiple comparisons test. (H, J, K) Representative MBP and NF200 immunofluorescence staining in peri-hematoma brain sections. n=3 mice/group. Scale bar: 100μm. Statistical analysis by the two-tailed t test. (I, L) Representative MBP and SMI-32 immunofluorescence staining in peri-hematoma brain sections. n=3-4 mice/group. Scale bar: 100 μm. Statistical analysis by the two-tailed t test. (M, P, Q) Representative immunofluorescence images and quantification of MBP and NF200 in the cervical spinal cord (n=3-4 mice/group). Scale bar: 100 μm. Statistical analysis by the two-tailed t test. ( N, O, R, S) Representative images and quantification of Iba-1 (microglia) and GFAP (astrocytes) immunofluorescence in the cervical spinal cord (n=3-4 mice/group). Scale bar: 100 μm. Statistical analysis by the Mann-Whitney test and the two-tailed t test, respectively. Data are presented as mean ± SEM. Significance levels are defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The membranes were blocked at room temperature in 5% non-fat milk for 1 hour, then incubated overnight at 4 □ with following primary antibodies: rabbit anti-NLRP3 (1:1000, immunoway, YM8024), rabbit anti-cleaved Caspase-1 p20 (1:1000, abmart, P79884R2M), rabbit anti-IL-1β (1:1000, MCE, HY-P80720), rabbit anti-ZO-1 (1:5000, proteintech, 21773-1-AP), rabbit anti-Occludin (1:5000, proteintech, 27260-1-AP), rabbit anti-β-tubulin (1:5000, affinity, AF7011), and rabbit anti-actin (1:5000, Beyotime, AF5006).

Techniques: Western Blot, Two Tailed Test, Immunofluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut Microbiota Dysbiosis-Mediated Gut NLRP3 Inflammasome Activation Exacerbates Corticospinal Tract Injury After Intracerebral Hemorrhage

doi: 10.64898/2026.02.23.707330

Figure Lengend Snippet: (A-B) Western blot analysis of NLRP3 inflammasome components (NLRP3, Caspase-1 p20, IL-1β) and tight junction proteins (ZO-1, Occludin) in brain tissue. β-Tubulin was used as the loading control (n=4 mice/group). Statistical analysis by the two-tailed t test. (C-D) ELISA quantification of serum levels of IL-1β (C) and Caspase-1 (D) from peripheral blood (n=4-5 and 5-6 mice/group, respectively). Statistical analysis by two-tailed t test with Welch’s correction and two-tailed t test, respectively. All data are presented as mean□±□SEM. Data were considered significant if *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The membranes were blocked at room temperature in 5% non-fat milk for 1 hour, then incubated overnight at 4 □ with following primary antibodies: rabbit anti-NLRP3 (1:1000, immunoway, YM8024), rabbit anti-cleaved Caspase-1 p20 (1:1000, abmart, P79884R2M), rabbit anti-IL-1β (1:1000, MCE, HY-P80720), rabbit anti-ZO-1 (1:5000, proteintech, 21773-1-AP), rabbit anti-Occludin (1:5000, proteintech, 27260-1-AP), rabbit anti-β-tubulin (1:5000, affinity, AF7011), and rabbit anti-actin (1:5000, Beyotime, AF5006).

Techniques: Western Blot, Control, Two Tailed Test, Enzyme-linked Immunosorbent Assay, IF-P

Journal: bioRxiv

Article Title: Gut Microbiota Dysbiosis-Mediated Gut NLRP3 Inflammasome Activation Exacerbates Corticospinal Tract Injury After Intracerebral Hemorrhage

doi: 10.64898/2026.02.23.707330

Figure Lengend Snippet: (A) Summary of the experimental procedures, timepoints, created by BioRender ( https://BioRender.com/dmwrlny ). (B-C) Western blot analysis of NLRP3 inflammasome components (NLRP3, Caspase-1 p20, IL-1β) and tight junction proteins (ZO-1, Occludin) in colon tissue (n=4 mice/group). β-Actin was used as the loading control. Statistical analysis by the Mann-Whitney test, except for the IL-1β protein data, was analyzed by the two-tailed t test. (D) Elisa analysis of serum Caspase-1 from peripheral blood. n=3-4 mice/group. Statistical analysis by two-tailed t test with Welch’s correction . (E) Elisa analysis of serum IL-1β from peripheral blood. n=6 mice/group. (F-G) Western blot analysis of NLRP3 inflammasome components (NLRP3, Caspase-1 p20, IL-1β) and BBB proteins (ZO-1, Occludin) in brain tissue in brain tissue. n=4 mice/group. β-tubulin was used as the loading control in Western blot analysis. Statistical analysis by two-tailed t test. Data are presented as mean ± SEM. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The membranes were blocked at room temperature in 5% non-fat milk for 1 hour, then incubated overnight at 4 □ with following primary antibodies: rabbit anti-NLRP3 (1:1000, immunoway, YM8024), rabbit anti-cleaved Caspase-1 p20 (1:1000, abmart, P79884R2M), rabbit anti-IL-1β (1:1000, MCE, HY-P80720), rabbit anti-ZO-1 (1:5000, proteintech, 21773-1-AP), rabbit anti-Occludin (1:5000, proteintech, 27260-1-AP), rabbit anti-β-tubulin (1:5000, affinity, AF7011), and rabbit anti-actin (1:5000, Beyotime, AF5006).

Techniques: Western Blot, Control, MANN-WHITNEY, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Gut Microbiota Dysbiosis-Mediated Gut NLRP3 Inflammasome Activation Exacerbates Corticospinal Tract Injury After Intracerebral Hemorrhage

doi: 10.64898/2026.02.23.707330

Figure Lengend Snippet: (A) Schematic of experimental design. (B-C) Colon NLRP3 inflammasome components (NLRP3, Caspase-1 p20, IL-1β) and gut barrier (ZO-1 and Occludin) were analyzed by Western blot. n=4 mice/group. Statistical analysis was performed using the one-way ANOVA test followed by Tukey’s post hoc test for multiple comparisons. (D-E) Representative footprints and the regularity Index from the CatWalk XT analysis, n=6 mice/group. Statistical analysis was performed using the two-way ANOVA test with Tukey’s multiple comparisons test. (F-G) Representative movement traces and quantification of total distance traveled at 1d, 3d, 7d, and 14 days post-ICH. n=4-5 mice/group. Statistical analysis was performed using the two-way ANOVA test followed by Tukey’s post hoc test for multiple comparisons. (H, J, K) MBP and NF200 immunofluorescence staining in peri-hematoma brain. n=4-6 mice/group. Scale bar:100 μm. One-way ANOVA test with Tukey’s multiple comparisons test was used for MBP, while Kruskal-Wallis test with Dunn’s multiple comparisons test was used for NF200. ( I, L ) MBP and SMI-32 immunofluorescence in peri-hematoma brain. n=5mice/group. Scale bar:100 μm. Statistical analysis by Welch’s ANOVA test with Games-Howell’s multiple comparisons test. (M, P, Q) MBP and NF200 immunofluorescence in the cervical spinal cord (n=4-6 mice/group). Scale bar: 100 μm. One-way ANOVA with Tukey’s multiple comparisons test was used for MBP and NF200. (N, O, R, S) Representative Iba-1 and GFAP immunofluorescence staining were performed on cervical spinal cord sections. n=3-5 mice/group. Scale bar:100 μm. One-way ANOVA with Tukey’s multiple comparisons test was used for Iba-1 and GFAP. (T-U) Western blot analysis of brain NLRP3, IL-1β, ZO-1 and Occludin. n=4 mice/group. One-way ANOVA with Tukey’s multiple comparisons test was used for these. β-tubulin or β-actin was used as the loading control in western blot analysis. All data are presented as meanLJ±LJSEM. *: AAV-GFP_FMT-ICH vs. AAV-NLRP3_FMT-ICH; #: AAV-GFP_FMT-ICH vs. AAV-GFP_Vehicle; *, # p < 0.05, **, ## p < 0.01, ***, ### p < 0.001.

Article Snippet: The membranes were blocked at room temperature in 5% non-fat milk for 1 hour, then incubated overnight at 4 □ with following primary antibodies: rabbit anti-NLRP3 (1:1000, immunoway, YM8024), rabbit anti-cleaved Caspase-1 p20 (1:1000, abmart, P79884R2M), rabbit anti-IL-1β (1:1000, MCE, HY-P80720), rabbit anti-ZO-1 (1:5000, proteintech, 21773-1-AP), rabbit anti-Occludin (1:5000, proteintech, 27260-1-AP), rabbit anti-β-tubulin (1:5000, affinity, AF7011), and rabbit anti-actin (1:5000, Beyotime, AF5006).

Techniques: Western Blot, Immunofluorescence, Staining, Control

Journal: Scientific reports

Article Title: Nrf2/HO-1 mediates the neuroprotective effect of mangiferin on early brain injury after subarachnoid hemorrhage by attenuating mitochondria-related apoptosis and neuroinflammation.

doi: 10.1038/s41598-017-12160-6

Figure Lengend Snippet: Figure 4. MF inhibits the activation of inflammasomes in rat brain tissues following SAH. Western blot analysis of the expression of NLRP3, ASC, and cleaved caspase-1 (p20) and the maturation of IL-1 β and IL-18 after SAH induction or MF treatment. n = 6 per group, #P < 0.05, ##P < 0.01, nsP > 0.05; *P < 0.05, **<0.01, compared with the Sham group.

Article Snippet: The membranes (PVDF) were subsequently blocked and incubated overnight at 4 °C with the following primary antibodies: anti-p-NF-κB p65 (1:500) (ab76302, Abcam, Cambridge, UK), anti-Bcl-2 (1:1000) (3498, Cell Signaling Technology, MA, USA), anti-Bax(1:2000)(ab32503, Abcam, Cambridge, UK), anti-cytochrome c (Cyto C) (1:1000) (11940, Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 (CC3) (1:1000) (9661, Cell Signaling Technology, MA, USA), anti-NLRP3 (1:1000) (15101, Cell Signaling Technology, MA, USA), anti-ASC (1:1000) (67824, Cell Signaling Technology, MA, USA), anti-caspase-1 p20 (1:1000) (ab207802, Abcam, Cambridge, UK), anti-IL-1β (1:1000) (ab2105, Abcam, Cambridge, UK), anti-IL-18 (1:1000) (ab71495, Abcam, Cambridge, UK), anti-Nrf2 (1:1000) (ab62352, Abcam, Cambridge, UK), anti-HO-1 (1:1000) (70081, Cell Signal Technology, MA, USA), anti-lamin B1 (1:2000) (ab133741, Abcam, Cambridge, UK) and anti-β-actin (1:2000) (SC-47778, Santa Cruz, CA, USA) blotted on the same membranes as a control.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Cell death & disease

Article Title: ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis.

doi: 10.1038/s41419-023-06237-6

Figure Lengend Snippet: Fig. 4 Effects of ChemR23 activation with RvE1 or C-9 on neuronal pyroptosis in CCH rats. A Representative transmission electron micrographs of hippocampal neurons in different treatment groups. Black arrowhead: membrane pores. Scale bar: 2 μm. n = 3 per group. B, C Representative double immunofluorescence staining of GSDMD/NeuN and NLRP3/NeuN in the rat hippocampus. Scale bar: 20 μm. n = 3 per group. D, E Quantitative statistics of GSDMD/NeuN or NLRP3/NeuN double-positive cells in rat hippocampus. n = 3 per group. F, G ELISA results of IL-1 and IL-18 analysis in the rat hippocampus. n = 4 per group. H, I Representative immunoblotting bands and the expression levels of NLRP3, GSDMD-N, Caspase-1 p20 and ASC in the rat hippocampus. n = 4 per group. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 vs. sham; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CCH.

Article Snippet: The membranes were then blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C with primary antibodies as follows: synaptophysin (SYN) (1:1000, A6344, ABclonal, Wuhan, China), postsynaptic density protein-95 (PSD95) (1:1000, A7889, ABclonal, Wuhan, China), NLRP3 (1:500, GB114320, Servicebio, Wuhan, USA), GSDMD (1:1000, GB114198, Servicebio, Wuhan, China), ASC (1:1000, A16672, ABclonal, Wuhan, China), caspase1-p20 (1:1000, A16792, ABclonal, Wuhan, China), Bcl-2 (1:1000, A0208, ABclonal, Wuhan, China), Bax (1:1000, A0207, ABclonal, Wuhan, China), LC3B (1:1000, A7198, ABclonal, Wuhan, China), SQSTM1/p62 (1:1000, A7758, ABclonal, Wuhan, China), Nrf2 (1:1000, GB113808, Servicebio, Wuhan, China), CMKLR1 (1:1000, No. 10325, Cayman, Michigan, USA), p-PI3K (1:1000, AP0854, ABclonal, Wuhan, China), PI3K (1:1000, A0982, ABclonal, Wuhan, China), p-AKT (1:1000, AP0140, ABclonal, Wuhan, China), AKT (1:1000, A17909, ABclonal, Wuhan, China) and β-actin (1:1000, 3700, CST, Massachusetts, USA).

Techniques: Activation Assay, Transmission Assay, Membrane, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Cell death & disease

Article Title: ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis.

doi: 10.1038/s41419-023-06237-6

Figure Lengend Snippet: Fig. 7 Effects of ChemR23 activation on pyroptosis in SH-SY5Y cells after hypoglycaemic and hypoxic stimulation. A Changes in cell viability with time under hypoglycaemic and hypoxic conditions. n = 4 per group. B, C Effects of different concentrations of RvE1 and C-9 on cell viability. n = 4 per group. D, E The proportion of Annexin V/PI double positive cells analysed by flow cytometry. n = 3 per group. F LDH levels in the cell culture supernatant. n = 3 per group. G–K Western blot analysis of NLRP3, GSDMD-N, Caspase-1 p20 and ASC. n = 3 per group. L, M Concentrations of IL-1β and IL-18 in the cell culture supernatant were measured by ELISA. n = 4 per group. (N) Representative immunofluorescence staining of NLRP3 (green) with the nuclei counterstained by DAPI. Scale bar: 50 μm. n = 3 per group. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 vs. Con; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HH; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. HH+RvE1; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. HH + C-9.

Article Snippet: The membranes were then blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C with primary antibodies as follows: synaptophysin (SYN) (1:1000, A6344, ABclonal, Wuhan, China), postsynaptic density protein-95 (PSD95) (1:1000, A7889, ABclonal, Wuhan, China), NLRP3 (1:500, GB114320, Servicebio, Wuhan, USA), GSDMD (1:1000, GB114198, Servicebio, Wuhan, China), ASC (1:1000, A16672, ABclonal, Wuhan, China), caspase1-p20 (1:1000, A16792, ABclonal, Wuhan, China), Bcl-2 (1:1000, A0208, ABclonal, Wuhan, China), Bax (1:1000, A0207, ABclonal, Wuhan, China), LC3B (1:1000, A7198, ABclonal, Wuhan, China), SQSTM1/p62 (1:1000, A7758, ABclonal, Wuhan, China), Nrf2 (1:1000, GB113808, Servicebio, Wuhan, China), CMKLR1 (1:1000, No. 10325, Cayman, Michigan, USA), p-PI3K (1:1000, AP0854, ABclonal, Wuhan, China), PI3K (1:1000, A0982, ABclonal, Wuhan, China), p-AKT (1:1000, AP0140, ABclonal, Wuhan, China), AKT (1:1000, A17909, ABclonal, Wuhan, China) and β-actin (1:1000, 3700, CST, Massachusetts, USA).

Techniques: Activation Assay, Cytometry, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell death & disease

Article Title: ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis.

doi: 10.1038/s41419-023-06237-6

Figure Lengend Snippet: Fig. 8 ChemR23 activation inhibited neuronal pyroptosis via the PI3K/AKT/Nrf2 signalling pathway in SH-SY5Y cells. A Scanning electron microscopy analysis showing the cell morphology in different treatment groups. n = 3 per group. B Representative immunofluorescence staining of NLRP3 (green) with the nuclei counterstained by DAPI. Scale bar: 50 μm. n = 3 per group. C LDH levels in the cell culture supernatant. n = 3 per group. D, E ELISA of IL-1β and IL-18 levels. n = 3 per group. F–J The levels of NLRP3, GSDMD-N, Caspase-1 p20 and ASC were evaluated by western blot. n = 3 per group. K–N The expression levels of p-PI3K/PI3K, p-AKT/AKT and Nrf2 were evaluated by western blot. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HH; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. HH + OE; N.S. means not significant vs. HH + OE.

Article Snippet: The membranes were then blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C with primary antibodies as follows: synaptophysin (SYN) (1:1000, A6344, ABclonal, Wuhan, China), postsynaptic density protein-95 (PSD95) (1:1000, A7889, ABclonal, Wuhan, China), NLRP3 (1:500, GB114320, Servicebio, Wuhan, USA), GSDMD (1:1000, GB114198, Servicebio, Wuhan, China), ASC (1:1000, A16672, ABclonal, Wuhan, China), caspase1-p20 (1:1000, A16792, ABclonal, Wuhan, China), Bcl-2 (1:1000, A0208, ABclonal, Wuhan, China), Bax (1:1000, A0207, ABclonal, Wuhan, China), LC3B (1:1000, A7198, ABclonal, Wuhan, China), SQSTM1/p62 (1:1000, A7758, ABclonal, Wuhan, China), Nrf2 (1:1000, GB113808, Servicebio, Wuhan, China), CMKLR1 (1:1000, No. 10325, Cayman, Michigan, USA), p-PI3K (1:1000, AP0854, ABclonal, Wuhan, China), PI3K (1:1000, A0982, ABclonal, Wuhan, China), p-AKT (1:1000, AP0140, ABclonal, Wuhan, China), AKT (1:1000, A17909, ABclonal, Wuhan, China) and β-actin (1:1000, 3700, CST, Massachusetts, USA).

Techniques: Activation Assay, Electron Microscopy, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing